The long term goals of the proposed research are to determine how the structures of Na+-dependent cotransport proteins affect their functional properties, and to identify the molecular mechanism of Na+-dependent cotransport, by studying cloned renal Na+-dependent cotransporters. The Na+-dependent transport or organic solutes is central to the function of the kidney in conserving essential nutrients that are filtered from the blood. The Na+-dependent organic solute cotransporters are predominantly found on the apical membranes of the cells of the proximal tubule. The properties of many Na+-dependent transporters of the kidney have been studied, but there is as yet no information on the mechanism of transport at the molecular level. With the development of recombinant DNA techniques, it is now possible to isolate cDNAs that code for renal Na+- dependent cotransport proteins and to study their function and structural characteristics in greater detail. It now appears that many Na+- dependent cotransporters are structurally related, and form families of proteins, including the SGLT family, related to the intestinal Na+/glucose cotransporter (SGLT1). The proposed research will focus on cloned renal transport proteins that are members of the SGLT family: the Na+/nucleoside cotransporter (SNST1), a putative transporter, RK-D, that was recently sequenced, and one of the renal Na+/glucose cotransporters, SGLT1. The first specific aim of this proposal is to examine the transport properties of two of the clones, SNST1 and RK-D, in expression systems including Xenopus oocytes, COS cells and Sf9 cells. A method of purifying plasma membranes containing the expressed clones will be developed in order to be able to do more precise kinetic determinations. The second specific aim is to determine the tissue distribution of SNST1 and RK-D by Northern and Western blotting. This information will help to determine the physiological role of these transporters in the body. The third specific aim is to examine the effects of post-translational modifications, such as phosphorylation and glycosylation, on the transport of SNST1 and RK-D. These modifications, particularly phosphorylation, may be important in regulating the function of these transporters. Finally, the fourth specific aim is to determine the regions of the transporters that are involved in conferring differences in substrate selectivity. The experiments in this final section will be done by constructing chimeric proteins between these structurally related transporters. These studies should provide fundamental information on the functional properties of these Na+-dependent transporters, and on the physiological role of these transporters in the kidney.